In vitro method for detecting avian intestinal dysbiosis

ABSTRACT

The invention pertains to an in vitro method for detecting avian intestinal dysbiosis, the method comprising determining the presence and/or level of isoleucyl-arginine (C12H25O3N5) or isomers thereof in avian sample material wherein the presence and/or an increased level of isoleucyl-arginine (C12H25O3N5) or isomers thereof in comparison to a non-affected control is indicative for avian intestinal dysbiosis.

FIELD OF THE INVENTION

The present invention relates to an in vitro method for detecting avian intestinal dysbiosis, the method comprising determining the presence and/or level of isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof in avian sample material, wherein the presence and/or an increased level of isoleucyl-arginine (C₁₂H₂₅O₃N₅) in comparison to a non-affected control is indicative for avian intestinal dysbiosis.

BACKGROUND OF THE INVENTION

Intestinal health is critically important for the welfare and performance of avian livestock animals.

The gastrointestinal tract (GIT) of avians hosts a complex microbial ecosystem containing trillions of microbes. These microbes are distributed throughout various niches performing diverse functions including the fermentation of foods. The microbial diversity within the GIT is a function of site of digestion, health status and age of the birds. Under normal conditions, the disease-inducing activity (dysbiosis) and disease-protective activity (probiosis) is perfectly balanced. However, even a slight disturbance in normal microbiota of the gut can lead to an imbalance of host-microbe relationships. This state of condition where microbial imbalance exerts adverse effects on the host is known as dysbiosis (dysbacteriosis).

Dysbiosis is detrimental to the host, leading to inflammation and mucosal tissue damage that predisposes them to pathological conditions like Clostridium perfringens infection or inflammatory bowel disease.

Ducatelle et al. in “Biomarkers for monitoring intestinal health in poultry: present status and future perspectives”, VETERINARY RESEARCH, vol. 49, no. 1, 8 May 2018 provide an overview about biomarkers related to intestinal health that are commonly known in the art, such as D-lactate in blood and liver and butyrate in cecal content/feces.

Clostridium perfringens is an ubiquitous pathogen that uses an arsenal of toxins to cause histotoxic and intestinal infections in animals and also in humans. C. perfringens is a Gram-positive, rod-shaped, spore forming, oxygen-tolerant anaerobe. Not all C. perfringens strains are virulent. As an animal pathogen, C. perfringens is responsible for several serious diseases including avian necrotic enteritis, which drains approximately US$6 billion/year from the global agricultural system [Wade, B., Keyburn, A. L. (2015), “The true cost of necrotic enteritis” World Poultry 31, 16-17].

Further, the disruption of the microbiota, for instance due to antibiotic treatment, favors growth of pathogenic bacteria, such as Salmonella (Santos R L. Pathobiology of salmonella, intestinal microbiota, and the host innate immune response. Front Immunol. 2014 May 26; 5:25), Campylobacter (Agunos A, Waddell L, Leger D, Taboada E. A systematic review characterizing on-farm sources of Campylobacter spp. for broiler chickens. PLoS One. 2014; 9(8):e104905. Published 2014 Aug. 29. doi:10.1371/journal.pone.0104905) Escherichia coli (colibacillosis: Yersinia enterocolitica (Mohammad M. Soltan Dallal, Michael P. Doyle, Maryam Rezadehbashi, Hossein Dabiri, Maryam Sanaei, Shabnam Modarresi, Rounak Bakhtiari, Kazem Sharifiy, Mahnaz Taremi, Mohammad R. Zali, M. K. Sharifi-Yazdi, Prevalence and antimicrobial resistance profiles of Salmonella serotypes, Campylobacter and Yersinia spp. isolated from retail chicken and beef, Tehran, Iran, Food Control, Volume 21, Issue 4, 2010, p 388-392)), and Pseudomonas (Devriese L A, Viaene N J, Demedts G. Pseudomonas aeruginosa infection on a broiler farm. Avian Pathol. 1975; 4(3):233-7).

In view of the above and in order to enable a timely and targeted intervention against avain intestinal dysbiosis, it was an urgent need to provide a fast and reliable, non-invasive ante mortem method for detecting dysbiosis in avian subjects or in avian populations.

SUMMARY OF THE INVENTION

The present invention provides an in vitro method for detecting avian intestinal dysbiosis, the method comprising determining the presence and/or level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in avian sample material, wherein the presence and/or an increased level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in comparison to a non-affected control is indicative for avian intestinal dysbiosis.

Further, the present invention pertains to the use of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof as markers for detecting avian intestinal dysbiosis, for deducting the necessity of nutritional or therapeutic interventions to be taken against avian intestinal dysbiosis, or for controlling the effectiveness of nutritional or therapeutic interventions taken against avian intestinal dysbiosis.

Finally, the present invention provides an in vitro mass-spectrometry-based method for detecting avian intestinal dysbiosis, the method comprising assaying avian sample material for isoleucyl-arginin or isomers thereof (C₁₂H₂₅O₃N₅, m/z=144.61, Mw=287.20), wherein the presence and/or an increased level of said biomarker indicates avian intestinal dysbiosis.

In the following, the crucial aspects of the present invention are described in detail.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have unexpectedly found that isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof constitute a diagnostic marker suitable for detecting avian intestinal dysbiosis.

Accordingly, the invention provides an in vitro method for detecting avian intestinal dysbiosis, the method comprising determining the presence and/or level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in avian sample material wherein the presence and/or an increased level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in comparison to a non-affected control is indicative for avian intestinal dysbiosis.

C₁₂H₂₅O₃N₅ is a dimeric amino acid occurring in the form of four possible isomers:

As each one of these isomers may serve as a biomarker for detecting avian intestinal dysbiosis, the term “isoleucyl-arginine” as used in the context of the present invention includes all four isomers depicted in the above.

As used in the context of the present invention, intestinal dysbiosis is a term for a microbial imbalance or maladaptation in the gastrointestinal tract, as defined in the above.

The non-affected control is a reference sample representing a proven intact gastrointestinal tract. As an example, a reference sample may be taken within an animal trial from an animal of a non-treated control, which was checked via pathology, histopathology and/or other measures to have no signs of dysbiosis.

The method of the present invention may be either applied for an individual avian subject or for a group or population of avians, e.g. for an avian population as occurring in livestock production.

The avian subject to be tested is preferably poultry. Preferred poultry according to the invention are chickens, turkeys, ducks and geese. The poultry can be optimized for producing young stock. This type of poultry is also referred to as parent and grandparent animals. Preferred parent and grandparent animals are, accordingly, (grand)parent broilers, (grand)parent ducks, (grand)parent turkeys and (grand)parent geese.

The poultry according to the invention can also be selected from fancy poultry and wild fowl. Preferred fancy poultry or wild fowl are peacocks, pheasants, partridges, guinea fowl, quails, capercailzies, goose, pigeons and swans. Further preferred poultry according to the invention are ostriches and parrots. Most preferred poultry according to the invention are broilers.

Preferably, the avian sample material is or comprises avian excremental material, in particular avian feces.

The intestinal sample material obtained from an individual avian may be selected from the group consisting of gut content samples, samples of bodily excrements and solutions or suspensions thereof; and from materials being contaminated with bodily excrements. The term “gut content” is to be understood as the content of the small intestine, the content of the large intestine and/or the content of the caecum. Methods for taking such gut content samples are known in the art.

As used in the context of the present invention, bodily excrements are fecal or cecal excrements. Materials being contaminated with bodily excrements are, for example, dust samples, swab samples, litter samples, liquid manure samples, fur samples, feather samples and skin samples.

In general, the term “litter” is to be understood as a mixture of animal excrements with the bedding material.

As used in the context of this embodiment, the term “litter samples” refers to excremental droppings from an individual animal. Further, in the context this embodiment, the term “liquid manure samples” refers to an excremental sample containing feces and urine from an individual animal.

Samples from individual animals can be taken either directly from the animal, e.g. with swabs. Alternatively and especially in case of single-housed animals, the sample material can be collected from the floor of the pen, cage or slat. The sample material has to be assignable to the investigated animal.

In one embodiment, the intestinal sample material used for determining whether or not an individual animal suffers from dysbiosis is feces.

For specific applications, it is also useful to analyze gut content samples, e.g. samples from the small intestine, samples from the large intestine and/or samples from the caecum.

In an alternative embodiment, the method is used for determining whether or not an avian population suffers from dysbiosis. In that case, the sample material is a pooled sample originating from the avian population to be tested. The avian sample material preferably is or comprises pooled avian feces deriving from an avian flock. The animal population preferably is an avian flock. The avian flock according to the invention is preferably poultry. Preferred poultry is as indicated in the before.

Accordingly, the method of the present invention is particularly suitable for determining the health status of an avian population via bulk testing. As used herein, the term “bulk testing” refers to a test method, wherein the sample material is a pooled sample of an animal population. A “pooled sample” in the context of this embodiment is to be understood as a composite sample from randomly selected separate samples, one sample taken with one or several moistened fabric swabs or pooled samples made up of separate samples of fresh samples taken at random from a number of sites in the house or space in which the animal population or the animal flock is kept. It may be necessary that the sample material is homogenized prior to sample analysis. Suitable homogenization techniques are known in the art.

As used in the context of the invention, the term “litter samples” refers to mixed excremental droppings in the pen, cage or slat. Further, in the context this embodiment, the term “liquid manure samples” refers to mixed excremental samples containing feces and urine.

These litter samples can, for example, be collected from an animal population using the overshoe method or using litter grabs at different places in the pen.

Boot swabs being sufficiently absorptive to soak up moisture are particularly suitable for collecting pooled animal samples. Tube gauze socks are also acceptable.

In case the animal population is kept in cages or slats, the excremental samples may be collected by a conveying belt.

Suitable sample volumes are, for example, 0.05 ml to 20 ml or 0.1 to 20 ml, in particular 0.2 to 10 ml, preferably 0.5 to 5 ml. Suitable sample masses are, for example 0.05 g to 20 g or 0.1 to 20 g, in particular 0.2 to 10 g, preferably 0.5 to 5 g.

The pooled samples reflect the amount of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) present in the animal population.

The isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof may be detected and/or quantified via LC-MS. As an alternative, isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof may be detected and/or quantified via an enzyme assay.

The isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof may also be detected and/or quantified via Liquid chromatography (LC) combined with pre- or post-column derivatization and with fluorescence or UV detection. Therefor, a wide variety of alternative pre- and post-column derivatization protocols may be used reagents such as ninhydridrin, o-phthalaldehyde (OPA), phenylisothiocyanate (PITC), 4-(dimethylamino)azobenzene-4′-sulfonyl (DABSYL) chloride, 5-(dimethylamino)naphthalene-1-sulfonyl (DANSYL) chloride, or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) involving either UV or (mostly) fluorescence detection.

In accordance with the above, the present invention is also directed to the use of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof as markers for detecting avian intestinal dysbiosis.

The present invention provides the abovementioned methods for detecting dysbiosis and for determining the extent thereof, respectively. This enables the farmer to make a qualified decision on whether or not measures for improving intestinal health are to be taken.

Accordingly, the methods according to the present invention may be used for deducting the necessity of nutritional or therapeutic interventions to be taken against avian intestinal dysbiosis, or, alternatively, for controlling the effectiveness of nutritional or therapeutic interventions taken against avian intestinal dysbiosis.

Measures against the development and/or against the progression of dysbiosis involve feeding or administering health-promoting substances, such as zootechnical feed additives, or therapeutic agents. The term “administering” or related terms includes oral administration. Oral administration may be via drinking water, oral gavage, aerosol spray or animal feed. The term “zootechnical feed additive” refers to any additive used to affect favorably the performance of animals in good health or used to affect favorably the environment. Examples for zootechnical feed additives are digestibility enhancers, i.e. substances which, when fed to animals, increase the digestibility of the diet, through action on target feed materials; gut flora stabilizers; micro-organisms or other chemically defined substances, which, when fed to animals, have a positive effect on the gut flora; or substances which favorably affect the environment. Preferably, the health-promoting substances are selected from the group consisting of probiotic agents, praebiotic agents, botanicals, organic/fatty acids, zeolithes, bacteriophages and bacteriolytic enzymes or any combinations thereof.

The present invention also pertains to a mass spectrometry—based method for detecting avian intestinal dysbiosis, the method comprising assaying avian sample material for isoleucyl-arginin or isomers thereof (C₁₂H₂₅O₃N₅, m/z=144.61, Mw=287.20), wherein the presence and/or an increased level of said biomarker indicates avian intestinal dysbiosis.

Preferably, the avian sample material is a pooled fecal sample.

Applications of the methods according to the invention are for example ((i) aiding in the diagnosis and/or prognosis of avian intestinal dysbiosis, (ii) monitoring the progress or reoccurrence of avian intestinal dysbiosis, or (iii) aiding in the evaluation of treatment efficacy for an animal population undergoing or contemplating treatment.

Applications of the methods according to the present invention in particular help to avoid loss in animal performance like weight gain and feed conversion.

In the following, the invention is illustrated by non-limiting examples and exemplifying embodiments.

EXAMPLES Methods 1. Extraction Protocol for Metabolic Fingerprinting of Broiler Feces

-   -   Weigh 100 mg dry weight feces in a 15 mL tube,     -   Add 2 mL of ice cold 80% MeOH,     -   Add 100 μL of 100 ng μL-1 valine-d8 (internal standard),     -   Vortex 1 min and rotate 2 min,     -   Centrifuge at 1000 rpm for 10 min (at room temperature),     -   Transfer the supernatant into a 15 mL tube,     -   Use an 1 mL syringe with needle to transfer the supernatant to a         0.45 μm PA filter,     -   Collect the filtrate in a 1.5 mL Eppendorf vial,     -   Dilute (1:3, i.e. one part filtrate, 2 parts H₂O) the filtrate         with ultra pure H₂O and vortex 15 s,     -   Transfer 125 μl filtrate to an LC-MS vial.

2. Liquid Chromatography

Chromatographic separation was achieved on a Accela UHPLC pumping system (Thermo Scientific) equipped with an Acquity HSS T3 column (150×2.1, 1.8 □m, Waters). The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, employed for gradient-based elution of the targeted compounds (Table 1). Chromatographic separation of these compounds was effectuated in 18 minutes, at a flow rate of 0.4 mL min-1 and column oven temperature of 45° C. Applied elution gradient program of LC-MS method with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B):

Time (min) Solvent A Solvent B 0 98 2 1.5 98 2 7 75 25 8 40 60 12 5 95 14 5 95 14.1 98 2 18 98 2

3. Mass Spectrometry

The used full-scan high-resolution Exactive™ Orbitrap mass spectrometer (Thermo Fisher Scientific) was equipped with a HESI-II source. Mass spectrometric analysis was performed in polarity switching mode, thereby alternating between positive and negative ionization mode from scan to scan. This allowed to obtain data about positive and negative ions during each single run. Accurate mass spectra were acquired in an m/z scan range of 50-800 Da, at a mass resolution of 100,000 FWHM. Other instrumental parameters are presented in Table 2. Optimization of these parameters was based on a standard mixture, containing the analytical standards of 115 polar metabolites.

Parameter Value Parameter Value Sheath gas flow (AU) 50 HESI-II positions 0/B/1 Auxiliary gas flow (AU) 25 Skimmer voltage (V) +/−20 Sweep gas flow (° C.) 5 Tube lens voltage (V) +/−60 Capillary temperature (° C.) 250 Capillary voltage (V) +/−90 Heater temperature (° C.) 350 Fragmentation HCD gas off Spray voltage (V) 5 AGC target Balanced

EXAMPLES

Broiler chickens were infected with C. perfringens. Additionally, coccidiose and dysbiosis was induced. Litter samples were collected for subsequent analysis from pens according to the following table:

Litter sample table for metabolomics analyses from infected, non-infected, infected non-pathogenic strains, coccidiosis and dysbiosis broiler chickens Infection Infec- Total BHI Infec- tion Sam- number (control) tion Non- Coc- ple of Un- Path- Path- cidi- Dysbiosis/ Trial origin samples infected ogen ogen osis Coccidiosis Batch 1 litter 30 3 21 6 — — Batch 2 litter 21 3 13 5 — — Batch 4 litter 19 6 7 6 Batch 5 litter 19 6  4 3 6 (field) (field)

Samples were extracted and analyzed via LC-MS as detailed in the materials and methods section above.

The trials and field studies revealed the metabolite C₁₂H₂₅O₃N₅ with a high accurate mass of 144.6056 m/z and a retention time (RD of 1.49 min as a marker for dysbiosis in broiler chickens. More specifically, this metabolite showed higher abundances in litter samples, obtained from dysbiosis birds. This metabolite was either absent or showed much lower abundances in samples from healthy birds and birds with NE.

The results are presented in the below. Three groups of birds were considered: (1) healthy birds, (2) dysbiosis birds and (3) birds suffering from necrotic enteritis.

Integrated peak areas (arbitrary units), as obtained for the metabolite biomarker in the various litter samples, categorized as either necrotic enteritis (NE), dysbiosis or healthy.

Marker Sample Category C₁₂H₂₅O₃N₅ 1 NE 1216377 2 NE 0 3 NE 2358412 4 NE 2391243 5 Dysbiosis 6065093 6 Dysbiosis 2869630 7 Dysbiosis 950729 8 Dysbiosis 3812383 9 Dysbiosis 4559121 10 Dysbiosis 1568876 11 Healthy 0 12 Healthy 693921 13 Healthy 603716 14 Healthy 0 15 Healthy 250469 16 Healthy 0 17 Healthy 288096 18 Healthy 1127064 19 Healthy 692537

Fragmentation Analysis

dd-MS²/dd-SIM

17,500 resolution

2 e5 AGC target

Max injection time of 80 ms

Isolation window of 2.0 m/z

NCE: 20; 35; 60

Minimum AGC target of 1 e4

Apex trigger 2 to 5 s

Dynamic exclusion 10.0 s

Fragmentation Spectrum of C₁₂H₂₅O₃N₅—Fragmented at an Energy of 38.33 eV

m/z-value of Absolute intensity Relative fragment (arbitrary units) intensity 55.0549 2977 0.47 57.05796 3132.2 0.49 60.04513 17421.6 2.75 60.05637 128141.2 20.22 69.07055 85461.4 13.48 70.0658 330818.8 52.19 71.04989 5921.9 0.93 72.045 17131.7 2.7 72.08137 20841.2 3.29 84.08128 4854.7 0.77 86.09691 633857.1 100 100.07606 11907.7 1.88 112.0873 12010.7 1.89 114.09162 4669.7 0.74 114.1028 3283.5 0.52 116.07072 95459.7 15.06 128.10699 169818.6 26.79 130.09735 35905.3 5.66

Based on the fragmentation profile, isoleucyl-arginine is the most likely structure of the C₁₂H₂₅O₃N₅. However, it cannot be excluded that the other diaminoacids (leucyl-arginine, arginyl-isoleucine, and arginyl-leucine) matches the true identity of the C₁₂H₂₅O₃N₅. 

1-10. (canceled)
 11. An in vitro method for detecting avian intestinal dysbiosis, comprising determining the presence and/or level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in avian sample material, wherein the presence and/or an increased level of isoleucyl-arginine (C₁₂H₂₅O₃N₅, Mw=287.20) or isomers thereof in comparison to a non-affected control animal is indicative for avian intestinal dysbiosis.
 12. The method of claim 11, wherein the avian sample material is, or comprises, avian excremental material.
 13. The method of claim 11, wherein the avian sample material is, or comprises, avian feces.
 14. The method of claim 11, wherein the avian sample material is, or comprises, pooled avian feces deriving from an avian flock.
 15. The method of claim 11, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via LC-MS.
 16. The method of claim 11, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via an enzyme assay.
 17. The method of claim 11, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via LC combined with pre- or post-column derivatization and fluorescence or UV detection.
 18. The method of claim 14, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via LC-MS.
 19. The method of claim 14, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via an enzyme assay.
 20. The method of claim 14, wherein the isoleucyl-arginine (C₁₂H₂₅O₃N₅) or isomers thereof are detected and/or quantified via LC combined with pre- or post-column derivatization and fluorescence or UV detection.
 21. The method of claim 11, further comprising treating animals exhibiting the presence of, and/or an increased level of, isoleucyl-arginine or isomers thereof in comparison to a non-affected control animal with nutritional or therapeutic interventions to treat or prevent avian intestinal dysbiosis.
 22. The method of claim 11, wherein animals exhibiting the presence of, and/or an increased level of, isoleucyl-arginine or isomers thereof in comparison to a non-affected control animal are administered digestibility enhancers; gut flora stabilizers; micro-organisms or other chemically defined substances, which, when fed to animals, have a positive effect on the gut flora.
 23. The method of claim 11, wherein animals exhibiting the presence of, and/or an increased level of, isoleucyl-arginine or isomers thereof in comparison to a control animal are administered probiotic agents, prebiotic agents, botanicals, organic/fatty acids, zeolites, bacteriophages or bacteriolytic enzymes or any combinations thereof. 